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1.
Mol Psychiatry ; 23(2): 323-334, 2018 02.
Article in English | MEDLINE | ID: mdl-28093569

ABSTRACT

Positron emission tomography (PET) imaging with radiotracers that target translocator protein 18 kDa (TSPO) has become a popular approach to assess putative neuroinflammatory processes and associated microglia activation in psychotic illnesses. It remains unclear, however, whether TSPO imaging can accurately capture low-grade inflammatory processes such as those present in schizophrenia and related disorders. Therefore, we evaluated the validity of TSPO as a disease-relevant marker of inflammation using a translational approach, which combined neurodevelopmental and neurodegenerative mouse models with PET imaging in patients with recent-onset schizophrenia and matched controls. Using an infection-mediated neurodevelopmental mouse model, we show that schizophrenia-relevant behavioral abnormalities and increased inflammatory cytokine expression are associated with reduced prefrontal TSPO levels. On the other hand, TSPO was markedly upregulated in a mouse model of acute neurodegeneration and reactive gliosis, which was induced by intrahippocampal injection of kainic acid. In both models, the changes in TSPO levels were not restricted to microglia but emerged in various cell types, including microglia, astrocytes and vascular endothelial cells. Human PET imaging using the second-generation TSPO radiotracer [11C]DPA-713 revealed a strong trend towards reduced TSPO binding in the middle frontal gyrus of patients with recent-onset schizophrenia, who were previously shown to display increased levels of inflammatory cytokines in peripheral and central tissues. Together, our findings challenge the common assumption that central low-grade inflammation in schizophrenia is mirrored by increased TSPO expression or ligand binding. Our study further underscores the need to interpret altered TSPO binding in schizophrenia with caution, especially when measures of TSPO are not complemented with other markers of inflammation. Unless more selective microglial markers are available for PET imaging, quantification of cytokines and other inflammatory biomarkers, along with their molecular signaling pathways, may be more accurate in attempts to characterize inflammatory profiles in schizophrenia and other mental disorders that lack robust reactive gliosis.


Subject(s)
Receptors, GABA/metabolism , Schizophrenia/metabolism , Adult , Animals , Astrocytes/metabolism , Biomarkers/blood , Disease Models, Animal , Female , Humans , Inflammation/metabolism , Male , Mice , Mice, Inbred C57BL , Microglia/metabolism , Neuroimmunomodulation/physiology , Positron-Emission Tomography/methods , Receptors, GABA/analysis , Schizophrenia/diagnostic imaging
2.
Philos Trans A Math Phys Eng Sci ; 374(2074)2016 Aug 13.
Article in English | MEDLINE | ID: mdl-27402928

ABSTRACT

Hysteresis is more than just an interesting oddity that occurs in materials with a first-order transition. It is a real obstacle on the path from existing laboratory-scale prototypes of magnetic refrigerators towards commercialization of this potentially disruptive cooling technology. Indeed, the reversibility of the magnetocaloric effect, being essential for magnetic heat pumps, strongly depends on the width of the thermal hysteresis and, therefore, it is necessary to understand the mechanisms causing hysteresis and to find solutions to minimize losses associated with thermal hysteresis in order to maximize the efficiency of magnetic cooling devices. In this work, we discuss the fundamental aspects that can contribute to thermal hysteresis and the strategies that we are developing to at least partially overcome the hysteresis problem in some selected classes of magnetocaloric materials with large application potential. In doing so, we refer to the most relevant classes of magnetic refrigerants La-Fe-Si-, Heusler- and Fe2P-type compounds.This article is part of the themed issue 'Taking the temperature of phase transitions in cool materials'.

3.
Neuroscience ; 161(2): 635-54, 2009 Jun 30.
Article in English | MEDLINE | ID: mdl-19332109

ABSTRACT

Converging evidence from pharmacological and molecular studies has led to the suggestion that inhibition of glycine transporter 1 (GlyT1) constitutes an effective means to boost N-methyl-d-aspartate receptor (NMDAR) activity by increasing the extra-cellular concentration of glycine in the vicinity of glutamatergic synapses. However, the precise extent and limitation of this approach to alter cognitive function, and therefore its potential as a treatment strategy against psychiatric conditions marked by cognitive impairments, remain to be fully examined. Here, we generated mutant mice lacking GlyT1 in the entire forebrain including neurons and glia. This conditional knockout system allows a more precise examination of GlyT1 downregulation in the brain on behavior and cognition. The mutation was highly effective in attenuating the motor-stimulating effect of acute NMDAR blockade by phencyclidine, although no appreciable elevation in NMDAR-mediated excitatory postsynaptic currents (EPSC) was observed in the hippocampus. Enhanced cognitive performance was observed in spatial working memory and object recognition memory while spatial reference memory and associative learning remained unaltered. These findings provide further credence for the potential cognitive enhancing effects of brain GlyT1 inhibition. At the same time, they indicated potential phenotypic differences when compared with other constitutive and conditional GlyT1 knockout lines, and highlighted the possibility of a functional divergence between the neuronal and glia subpopulations of GlyT1 in the regulation of learning and memory processes. The relevance of this distinction to the design of future GlyT1 blockers as therapeutic tools in the treatment of cognitive disorders remains to be further investigated.


Subject(s)
Glycine Plasma Membrane Transport Proteins/genetics , Memory , Prosencephalon/physiology , Receptors, N-Methyl-D-Aspartate/antagonists & inhibitors , Amphetamine/pharmacology , Animals , Down-Regulation , Excitatory Postsynaptic Potentials , Female , Glycine/metabolism , Glycine Plasma Membrane Transport Proteins/biosynthesis , Hippocampus/physiology , Learning , Male , Mice , Mice, Knockout , Motor Activity/drug effects , Mutation , Phencyclidine/pharmacology , Prosencephalon/drug effects , Prosencephalon/metabolism , Psychomotor Performance , Receptors, N-Methyl-D-Aspartate/biosynthesis , Recognition, Psychology , Synaptic Transmission
4.
Proc Natl Acad Sci U S A ; 102(47): 17154-9, 2005 Nov 22.
Article in English | MEDLINE | ID: mdl-16284244

ABSTRACT

Overactivity of the dopaminergic system in the brain is considered to be a contributing factor to the development and symptomatology of schizophrenia. Therefore, the GABAergic control of dopamine functions was assessed by disrupting the gene encoding the alpha3 subunit of the GABA(A) receptor. alpha3 knockout (alpha3KO) mice exhibited neither an obvious developmental defect nor apparent morphological brain abnormalities, and there was no evidence for compensatory up-regulation of other major GABA(A)-receptor subunits. Anxiety-related behavior in the elevated-plus-maze test was undisturbed, and the anxiolytic-like effect of diazepam, which is mediated by alpha2-containing GABA(A) receptors, was preserved. As a result of the loss of alpha3 GABA(A) receptors, the GABA-induced whole-cell current recorded from midbrain dopamine neurons was significantly reduced. Spontaneous locomotor activity was slightly elevated in alpha3KO mice. Most notably, prepulse inhibition of the acoustic startle reflex was markedly attenuated in the alpha3KO mice, pointing to a deficit in sensorimotor information processing. This deficit was completely normalized by treatment with the antipsychotic D2-receptor antagonist haloperidol. The amphetamine-induced hyperlocomotion was not altered in alpha3KO mice compared with WT mice. These results suggest that the absence of alpha3-subunit-containing GABA(A) receptors induces a hyperdopaminergic phenotype, including a severe deficit in sensorimotor gating, a common feature among psychiatric conditions, including schizophrenia. Hence, agonists acting at alpha3-containing GABA(A) receptors may constitute an avenue for an effective treatment of sensorimotor-gating deficits in various psychiatric conditions.


Subject(s)
Dopamine/physiology , Ion Channel Gating/genetics , Motor Activity/genetics , Protein Subunits/deficiency , Receptors, GABA-A/deficiency , Schizophrenia/genetics , Schizophrenia/physiopathology , Amphetamine/pharmacology , Animals , Anti-Anxiety Agents/pharmacology , Diazepam/pharmacology , Disease Models, Animal , Electrophysiology , GABA Modulators/pharmacology , Gene Targeting , Haloperidol/pharmacology , Immunohistochemistry , Ion Channel Gating/physiology , Mice , Mice, Knockout , Motor Activity/drug effects , Motor Activity/physiology , Protein Subunits/genetics , Protein Subunits/physiology , Receptors, GABA-A/genetics , Receptors, GABA-A/physiology , Schizophrenia/drug therapy
5.
Proc Natl Acad Sci U S A ; 99(13): 8980-5, 2002 Jun 25.
Article in English | MEDLINE | ID: mdl-12084936

ABSTRACT

The heterogeneity of gamma-aminobutyric acid type A (GABA(A)) receptors contributes to the diversity of neuronal inhibition in the regulation of information processing. Although most GABA(A) receptors are located synaptically, the small population of alpha5GABA(A) receptors is largely expressed extrasynaptically. To clarify the role of the alpha5GABA(A) receptors in the control of behavior, a histidine-to-arginine point mutation was introduced in position 105 of the murine alpha5 subunit gene, which rendered the alpha5GABA(A) receptors diazepam-insensitive. Apart from an incomplete muscle relaxing effect, neither the sedative, anticonvulsant, nor anxiolytic-like activity of diazepam was impaired in alpha5(H105R) mice. However, in hippocampal pyramidal cells, the point mutation resulted in a selective reduction of alpha5GABA(A) receptors, which altered the drug-independent behavior. In line with the role of the hippocampus in certain forms of associative learning, trace fear conditioning, but not delay conditioning or contextual conditioning, was facilitated in the mutant mice. Trace fear conditioning differs from delay conditioning in that the conditioned and unconditioned stimulus are separated by a time interval. Thus, the largely extrasynaptic alpha5GABA(A) receptors in hippocampal pyramidal cells are implicated as control elements of the temporal association of threat cues in trace fear conditioning.


Subject(s)
Conditioning, Classical , Fear , Hippocampus/physiology , Receptors, GABA-A/physiology , Animals , Behavior, Animal , Immunohistochemistry , In Vitro Techniques , Long-Term Potentiation , Mice , Point Mutation , Receptors, GABA-A/drug effects , Receptors, GABA-A/genetics
6.
Life Sci ; 68(19-20): 2297-300, 2001 Apr 06.
Article in English | MEDLINE | ID: mdl-11358339

ABSTRACT

The slow component of GABAergic inhibition in the brain is mediated by the metabotropic GABA(B)-receptors. Most if not all GABA(B)-receptors are heterodimers of GABA(B)R1 (GBR1) and GABA(B)R2 (GBR2) proteins. Distinctive receptor isoforms are based on the presence of two GBR1 splice variants termed GBR1a and GBR1b. Both were found to be associated with GBR2 suggesting that the isoforms GBR1a/GBR2 and GBR1b/GBR2 represent the vast majority of GABA(B)-receptors in the brain. The two isoforms differed strikingly in their pattern of expression on the regional, cellular and subcellular level. These results point to distinct funcional roles of the two receptor isoforms.


Subject(s)
Brain/metabolism , Receptors, GABA-B/metabolism , Alternative Splicing , Animals , Brain Chemistry , Immunoenzyme Techniques , Molecular Structure , Protein Isoforms , RNA, Messenger/metabolism , Rats , Receptors, GABA-B/analysis , Receptors, GABA-B/genetics
7.
Biomaterials ; 22(9): 1005-12, 2001 May.
Article in English | MEDLINE | ID: mdl-11311004

ABSTRACT

The natural bone substitute Bio-Oss is used in periodontal and maxillofacial surgery to fill bone defects and permit reossification. Recent reports have suggested the presence of TGFbeta and of substantial amounts of protein in Bio-Oss and have questioned its position as a biologically inert material and its safety in clinical applications (Hönig et al., Plast Reconstr Surg 1999;103:1324; Schwartz et al., J Periodontol 2000;71:1258). Bio-Oss was therefore subjected to a detailed biochemical, histochemical and biophysical analysis. In three different types of extracts of Bio-Oss no evidence for the presence of protein based on SDS-PAGE and silver staining was detected. In addition, as shown by Western blotting, there was no immunochemical evidence for the presence of the potential growth-inducing factor TGFbeta. Furthermore, micropolished sections of Bio-Oss failed to be stained with McNeal's Tetrachrome as did microtome sections treated with Goldner's Trichrome. However, Bio-Oss was strongly stained with the protein dye Coomassie blue. This staining was virtually irreversible and is attributed to the carbonate content of Bio-Oss which was detected by thermogravimetry-mass spectrometry. Thus, within the limits of the assay conditions, Bio-Oss does not contain protein material to a measurable extent.


Subject(s)
Bone Substitutes/chemistry , Carbonates/analysis , Minerals/chemistry , Transforming Growth Factor beta/analysis , Blotting, Western , Electrophoresis, Polyacrylamide Gel , Indicators and Reagents , Microscopy/methods , Rosaniline Dyes
8.
Science ; 290(5489): 131-4, 2000 Oct 06.
Article in English | MEDLINE | ID: mdl-11021797

ABSTRACT

Benzodiazepine tranquilizers are used in the treatment of anxiety disorders. To identify the molecular and neuronal target mediating the anxiolytic action of benzodiazepines, we generated and analyzed two mouse lines in which the alpha2 or alpha3 GABAA (gamma-aminobutyric acid type A) receptors, respectively, were rendered insensitive to diazepam by a knock-in point mutation. The anxiolytic action of diazepam was absent in mice with the alpha2(H101R) point mutation but present in mice with the alpha3(H126R) point mutation. These findings indicate that the anxiolytic effect of benzodiazepine drugs is mediated by alpha2 GABAA receptors, which are largely expressed in the limbic system, but not by alpha3 GABAA receptors, which predominate in the reticular activating system.


Subject(s)
Anti-Anxiety Agents/pharmacology , Diazepam/pharmacology , Receptors, GABA-A/metabolism , Animals , Anti-Anxiety Agents/metabolism , Behavior, Animal/drug effects , Binding Sites , Brain/drug effects , Brain/metabolism , Cells, Cultured , Diazepam/metabolism , Dose-Response Relationship, Drug , Female , Gene Targeting , Hippocampus/cytology , Membrane Potentials/drug effects , Mice , Patch-Clamp Techniques , Phenobarbital/pharmacology , Point Mutation , Pyramidal Cells/drug effects , Pyramidal Cells/physiology , Receptors, GABA-A/chemistry , Receptors, GABA-A/genetics , Synaptic Transmission , gamma-Aminobutyric Acid/pharmacology
9.
J Physiol ; 527 Pt 1: 11-31, 2000 Aug 15.
Article in English | MEDLINE | ID: mdl-10944167

ABSTRACT

1. The aim of this study was to define the biophysical properties contributed by the gamma2 subunit to native single GABAA receptors. 2. Single-channel activity was recorded from neurones of wild-type (gamma2+/+) mice and compared with that from mice which were heterozygous (gamma2+/-) or homozygous (gamma2-/-) for a targeted disruption in the gamma2 subunit gene of the GABAA receptor. Unitary currents were evoked by low concentrations of GABA (0.5-5 microM) in membrane patches from acutely isolated dorsal root ganglion (DRG) neurones (postnatal day 0) and by 1 microM GABA in patches from embryonic hippocampal neurones which were cultured for up to 3 weeks. 3. GABAA receptors from DRG and hippocampal neurones of gamma2+/+ and gamma2+/- mice displayed predominantly a conductance state of 28 pS and less frequently 18 and 12 pS states. In gamma2-/- mice, conductance states mainly of 12 pS and less frequently of 24 pS were found. 4. The mean open duration of the 28 pS state in gamma2+/+ GABAA receptors (1.5-2.6 ms) was substantially longer than for the 12 pS state of gamma2-/- GABAA receptors (0.9-1.2 ms) at all GABA concentrations. For gamma2+/+ and gamma2-/- channels, the mean open duration was increased at higher GABA concentrations. 5. Open duration frequency distributions of 28 and 12 pS receptors revealed the existence of at least three exponential components. Components with short mean durations declined and components with long mean durations increased in relative frequency at higher GABA concentration indicating at least two binding sites of GABA per 28 and 12 pS receptor. 6. Shut time frequency distributions revealed at least four exponential components of which two were identified as intraburst components in 28 pS and one in 12 pS GABAA receptors. 7. The mean burst duration and the mean number of openings per burst increased in 28 and 12 pS GABAA receptors with increasing GABA concentration. At least two burst types were identified: simple bursts consisting of single openings and complex bursts of five to six openings in 28 pS but only two to three openings in 12 pS GABAA receptors. 8. We conclude that the gamma2 subunit enhances the efficacy of GABA by determining open conformations of high conductance and long lifetime, and by prolonging the time receptors remain in the activated bursting state.


Subject(s)
Ion Channels/metabolism , Neurons/physiology , Receptors, GABA-A/chemistry , Receptors, GABA-A/physiology , Animals , Animals, Newborn , Cells, Cultured , Electric Conductivity , Ganglia, Spinal/metabolism , Ganglia, Spinal/physiology , Hippocampus/metabolism , Hippocampus/physiology , Ion Channel Gating , Kinetics , Mice , Mice, Inbred C57BL , Mice, Knockout , Neurons/metabolism , Patch-Clamp Techniques , Protein Structure, Quaternary , Receptors, GABA-A/genetics , gamma-Aminobutyric Acid/pharmacology
10.
J Pharmacol Exp Ther ; 294(1): 73-9, 2000 Jul.
Article in English | MEDLINE | ID: mdl-10871297

ABSTRACT

Organic anion-transporting polypeptides (Oatps) are a rapidly growing gene family of polyspecific membrane transporters. In rat brain, Oatp1 (gene symbol Slc21a1) and Oatp2 (Slc21a5) are localized at the apical and basolateral domains, respectively, of the choroid plexus epithelium. Furthermore, Oatp2 is strongly expressed at the rat blood-brain barrier (BBB). This study localizes the human OATP (now called OATP-A; SLC21A3) at the BBB in humans. Furthermore, with the Xenopus laevis oocyte system the delta-opioid receptor agonists [D-penicillamine(2,5)]enkephalin (DPDPE) and deltorphin II were identified as new transport substrates of OATP-A. This OATP-A-mediated DPDPE and deltorphin II transport exhibited apparent K(m) values of approximately 202 and 330 microM, respectively, and OATP-A-mediated deltorphin II transport was inhibited by the mu-opioid receptor agonist Tyr-D-Ala-Gly-N-methyl-Phe-glycinol, the endogenous peptide Leu-enkephalin, and the opiate antagonists naloxone and naltrindole. DPDPE also was transported by rat Oatp1 (K(m) approximately 48 microM) and Oatp2 (K(m) approximately 19 microM), whereas deltorphin II was only transported by Oatp1 (K(m) approximately 137 microM). These results demonstrate that OATP-A can mediate transport of the analgesic opioid peptides DPDPE and deltorphin II across the human BBB. Furthermore, because rat Oatp1 and Oatp2 exhibit similar but not identical transport activities as OATP-A, the results generally indicate that members of the Oatp/OATP gene family of membrane transporters play an important role in carrier-mediated transport of opioid peptides across the BBB and blood-cerebrospinal fluid barrier of the mammalian brain.


Subject(s)
Blood-Brain Barrier , Carrier Proteins/physiology , Opioid Peptides/pharmacokinetics , Aged , Animals , Anion Transport Proteins , Biological Transport , Carrier Proteins/analysis , Enkephalin, D-Penicillamine (2,5)-/pharmacokinetics , Humans , Male , Molecular Weight , Oligopeptides/pharmacokinetics , Rats
11.
J Neurosci ; 20(8): 2758-65, 2000 Apr 15.
Article in English | MEDLINE | ID: mdl-10751426

ABSTRACT

Anxiety is a common psychiatric illness often treated by benzodiazepines (BZs). BZs, such as Valium, bind to the alpha subunit of the pentameric GABA(A) receptor and increase inhibition in the CNS. There is considerable evidence for abnormal GABA(A) receptor function in anxiety, and a significant proportion of anxiety patients has a reduced sensitivity to BZs. Here, we show that serotonin(1A) (5-HT(1A)) receptor knock-out mice display BZ-resistant anxiety. Consistent with this finding, binding of both BZ and non-BZ GABA(A) receptor ligands were reduced and GABAergic inhibition was impaired in mutant mice. These changes were reflected by abnormal alpha subunit expression in the amygdala and hippocampus, two important limbic regions involved in fear and anxiety. These data suggest a pathological pathway, initiated by a 5-HT(1A) receptor deficit, leading to abnormalities in GABA(A) receptor composition and level, which in turn result in BZ-insensitivity and anxiety. This model mechanistically links together the 5-HT and GABA systems, which both have been implicated in anxiety. A related mechanism may underlie reduced BZ sensitivity in certain forms of anxiety.


Subject(s)
Amygdala/drug effects , Anti-Anxiety Agents/pharmacology , Anxiety/genetics , Benzodiazepines/pharmacology , Hippocampus/drug effects , Receptors, GABA-A/metabolism , Receptors, Serotonin/genetics , Amygdala/metabolism , Animals , Anxiety/drug therapy , Anxiety/metabolism , Down-Regulation , Drug Resistance , Gene Silencing , Hippocampus/metabolism , Mice , Mice, Knockout , Receptors, GABA-A/drug effects , Receptors, Serotonin, 5-HT1
12.
Nature ; 401(6755): 796-800, 1999 Oct 21.
Article in English | MEDLINE | ID: mdl-10548105

ABSTRACT

GABA(A) (gamma-aminobutyric acid(A)) receptors are molecular substrates for the regulation of vigilance, anxiety, muscle tension, epileptogenic activity and memory functions, which is evident from the spectrum of actions elicited by clinically effective drugs acting at their modulatory benzodiazepine-binding site. Here we show, by introducing a histidine-to-arginine point mutation at position 101 of the murine alpha1-subunit gene, that alpha1-type GABA(A) receptors, which are mainly expressed in cortical areas and thalamus, are rendered insensitive to allosteric modulation by benzodiazepine-site ligands, whilst regulation by the physiological neurotransmitter gamma-aminobutyric acid is preserved. alpha1(H101R) mice failed to show the sedative, amnesic and partly the anticonvulsant action of diazepam. In contrast, the anxiolytic-like, myorelaxant, motor-impairing and ethanol-potentiating effects were fully retained, and are attributed to the nonmutated GABA(A) receptors found in the limbic system (alpha2, alpha5), in monoaminergic neurons (alpha3) and in motoneurons (alpha2, alpha5). Thus, benzodiazepine-induced behavioural responses are mediated by specific GABA(A) receptor subtypes in distinct neuronal circuits, which is of interest for drug design.


Subject(s)
Benzodiazepines/pharmacology , Diazepam/pharmacology , GABA Modulators/pharmacology , Receptors, GABA-A/drug effects , Animals , Anti-Anxiety Agents/pharmacology , Anticonvulsants/pharmacology , Arginine/metabolism , Benzodiazepines/metabolism , Drug Design , Histidine/metabolism , Hypnotics and Sedatives/pharmacology , Memory/drug effects , Mice , Mice, Inbred C57BL , Muscle Relaxants, Central/pharmacology , Point Mutation , Receptors, GABA-A/genetics , Receptors, GABA-A/metabolism
13.
Proc Natl Acad Sci U S A ; 96(22): 12860-5, 1999 Oct 26.
Article in English | MEDLINE | ID: mdl-10536013

ABSTRACT

Synaptic localization of gamma-aminobutyric acid type A (GABA(A)) receptors is a prerequisite for synaptic inhibitory function, but the mechanism by which different receptor subtypes are localized to postsynaptic sites is poorly understood. The gamma2 subunit and the postsynaptic clustering protein gephyrin are required for synaptic localization and function of major GABA(A) receptor subtypes. We now show that transgenic overexpression of the gamma3 subunit in gamma2 subunit-deficient mice restores benzodiazepine binding sites, benzodiazepine-modulated whole cell currents, and postsynaptic miniature currents, suggesting the formation of functional, postsynaptic receptors. Moreover, the gamma3 subunit can substitute for gamma2 in the formation of GABA(A) receptors that are synaptically clustered and colocalized with gephyrin in vivo. These clusters were formed even in brain regions devoid of endogenous gamma3 subunit, indicating that the factors present for clustering of gamma2 subunit-containing receptors are sufficient to cluster gamma3 subunit-containing receptors. The GABA(A) receptor and gephyrin-clustering properties of the ectopic gamma3 subunit were also observed for the endogenous gamma3 subunit, but only in the absence of the gamma2 subunit, suggesting that the gamma3 subunit is at a competitive disadvantage with the gamma2 subunit for clustering of postsynaptic GABA(A) receptors in wild-type mice.


Subject(s)
Receptors, GABA-A/metabolism , Synapses/metabolism , Animals , Base Sequence , Benzodiazepines/metabolism , Binding Sites , DNA Primers , Immunohistochemistry , Mice , Mice, Transgenic , Microscopy, Confocal , Receptors, GABA-A/chemistry
14.
Eur J Biochem ; 265(1): 189-94, 1999 Oct 01.
Article in English | MEDLINE | ID: mdl-10491173

ABSTRACT

Tritiated meta-sulfonate benzene diazonium ([3H]MSBD), a molecule structurally related to 4-aminobutyrate (GABA), which presents a reactivity toward nucleophilic amino acid residues, was synthesized to investigate the GABA binding site on the GABAA receptor. Irreversible labeling reactions using [3H]MSBD were performed on purified GABAA receptors isolated from cow brain membranes and labeled receptors were analyzed by SDS/PAGE. [3H]MSBD was found to be specifically incorporated into proteins in the 45-60 kDa molecular mass range which were identified as alpha1 subunits and beta2/beta3 subunits by immunoprecipitation with subunit-specific antibodies. The specific immunoprecipitation of alpha and beta subunits confirms that binding of [3H]MSBD occurs at the boundary of these subunits. These labeling results confirm the involvement of nucleophilic residues from the beta subunit but reveal also the contribution of yet unidentified nucleophilic residues on the alpha subunit for the GABA binding site.


Subject(s)
Diazonium Compounds/metabolism , GABA Antagonists/metabolism , Receptors, GABA-A/metabolism , gamma-Aminobutyric Acid/analogs & derivatives , Animals , Binding Sites , Cattle , Diazonium Compounds/chemistry , Isotope Labeling , Molecular Probes , Precipitin Tests , Receptors, GABA-A/immunology , Tritium
15.
J Biol Chem ; 274(38): 27323-30, 1999 Sep 17.
Article in English | MEDLINE | ID: mdl-10480953

ABSTRACT

The subunit architecture of gamma-aminobutyric acid, type B (GABA(B)), receptors in situ is largely unknown. The GABA(B) receptor variants, characterized by the constituents GBR1a and GBR1b, were therefore analyzed with regard to their subunit composition as well as their regional and subcellular distribution in situ. The analysis was based on the use of antisera recognizing selectively GBR1a, GBR1b, and GBR2. Following their solubilization, GBR1a and GBR1b were both found by immunoprecipitation to occur as heterodimers associated with GBR2. Furthermore, monomers of GBR1a, GBR1b, or GBR2 were not detectable, suggesting that practically all GABA(B) receptors are heterodimers in situ. Finally, there was no evidence for an association of GBR1a with GBR1b indicating that these two constituents represent two different receptor populations. A size determination of solubilized GABA(B) receptors by sucrose density centrifugation revealed two distinct peaks of which one corresponded to dimeric receptors, and the higher molecular weight peak pointed to the presence of yet unknown receptor-associated proteins. The distribution and relative abundance of GBR2 immunoreactivity corresponded in all brain regions to that of the sum of GBR1a and GBR1b, supporting the view that most if not all GBR1 proteins are associated with GBR2. However, GBR1a was present preferentially at postsynaptic densities, whereas GBR1b may be mainly attributed to presynaptic or extrasynaptic sites. Thus, GBR1a and GBR1b are both associated with GBR2 to form heterodimers at mainly different subcellular locations where they are expected to subserve different functions.


Subject(s)
Alternative Splicing , Receptors, GABA-B/genetics , Receptors, GABA-B/metabolism , Amino Acid Sequence , Animals , Centrifugation, Density Gradient , Male , Molecular Sequence Data , Rats , Rats, Sprague-Dawley , Solubility
16.
Nat Neurosci ; 2(9): 833-9, 1999 Sep.
Article in English | MEDLINE | ID: mdl-10461223

ABSTRACT

Patients with panic disorders show a deficit of GABAA receptors in the hippocampus, parahippocampus and orbitofrontal cortex. Synaptic clustering of GABAA receptors in mice heterozygous for the gamma2 subunit was reduced, mainly in hippocampus and cerebral cortex. The gamma2 +/- mice showed enhanced behavioral inhibition toward natural aversive stimuli and heightened responsiveness in trace fear conditioning and ambiguous cue discrimination learning. Implicit and spatial memory as well as long-term potentiation in hippocampus were unchanged. Thus gamma2 +/- mice represent a model of anxiety characterized by harm avoidance behavior and an explicit memory bias for threat cues, resulting in heightened sensitivity to negative associations. This model implicates GABAA-receptor dysfunction in patients as a causal predisposition to anxiety disorders.


Subject(s)
Anxiety/genetics , Anxiety/physiopathology , Cues , Hippocampus/physiology , Memory/physiology , Neurons/physiology , Receptors, GABA-A/physiology , Animals , Anxiety/psychology , Anxiety Disorders/genetics , Anxiety Disorders/physiopathology , Anxiety Disorders/psychology , Avoidance Learning/physiology , Conditioning, Operant , Fear , Heterozygote , Hippocampus/physiopathology , Maze Learning/physiology , Mice , Mice, Inbred C57BL , Mice, Inbred Strains , Neurons/drug effects , Patch-Clamp Techniques , Receptors, GABA-A/genetics , gamma-Aminobutyric Acid/pharmacology
17.
Neuropharmacology ; 38(2): 233-42, 1999 Feb.
Article in English | MEDLINE | ID: mdl-10218864

ABSTRACT

Activation of NMDA receptors requires the presence of glycine as a coagonist which binds to a site that is allosterically linked to the glutamate binding site. To identify the protein constituents of the glycine binding site in situ the photoaffinity label [3H]CGP 61594 was synthesized. In reversible binding assays using crude rat brain membranes, [3H]CGP 61594 labeled with high affinity (K(D) = 23 nM) the glycine site of the NMDA receptor. This was evident from the Scatchard analysis, the displacing potencies of various glycine site ligands and the allosteric modulation of [3H]CGP 61594 binding by ligands of the glutamate and polyamine sites. Electrophysiological experiments in a neocortical slice preparation identified CGP 61594 as a glycine antagonist. Upon UV-irradiation, a protein band of 115 kDa was specifically photolabeled by [3H]CGP 61594 in brain membrane preparations. The photolabeled protein was identified as the NR1 subunit of the NMDA receptor by NR1 subunit-specific immunoaffinity chromatography. Thus, [3H]CGP 61594 is the first photoaffinity label for the glycine site of NMDA receptors. It will serve as a tool for the identification of structural elements that are involved in the formation of the glycine binding domain of NMDA receptors in situ and will thereby complement the mutational analysis of recombinant receptors.


Subject(s)
Azides/pharmacology , Azides/pharmacokinetics , Brain/physiology , Excitatory Amino Acid Agonists/pharmacokinetics , Glycine/metabolism , Quinolines/pharmacology , Quinolines/pharmacokinetics , Receptors, N-Methyl-D-Aspartate/metabolism , Affinity Labels , Animals , Azides/chemical synthesis , Binding Sites , Binding, Competitive , Brain/drug effects , Cell Membrane/metabolism , Cerebral Cortex/physiology , Electrophysiology , Evoked Potentials/drug effects , Excitatory Amino Acid Agonists/pharmacology , Hippocampus/physiology , In Vitro Techniques , Kinetics , Ligands , Male , N-Methylaspartate/pharmacology , Neocortex/physiology , Quinolines/chemical synthesis , Rats , Rats, Sprague-Dawley , Serine/pharmacology , Tritium
18.
Eur J Neurosci ; 11(3): 761-8, 1999 Mar.
Article in English | MEDLINE | ID: mdl-10103070

ABSTRACT

GABAB (gamma-aminobutyric acid)-receptors have been implicated in central nervous system (CNS) functions, e.g. cognition and pain perception, and dysfunctions including spasticity and absence epilepsy. To permit an analysis of the two known GABAB-receptor splice variants GABAB-R1a (GB1a) and GABAB-R1b (GB1b), their distribution pattern has been differentiated in the rat brain, using Western blotting and immunohistochemistry with isoform-specific antisera. During postnatal maturation, the expression of the two splice variants was differentially regulated with GB1a being preponderant at birth. In adult brain, GB1b-immunoreactivity (-IR) was predominant, and the two isoforms largely accounted for the pattern of GABAB-receptor binding sites in the brain. Receptor heterogeneity was pronounced in the hippocampus, where both isoforms occurred in CA1, but only GB1b in CA3. Similarly, in the cerebellum, GB1b was exclusively found in Purkinje cells in a zebrin-like pattern. The staining was most pronounced in Purkinje cell dendrites and spines. Using electron microscopy, over 80% of the spine profiles in which a synaptic contact with a parallel fibre was visible contained GB1b-IR at extrasynaptic sites. This subcellular localization is unrelated to GABAergic inputs, indicating that the role of GABAB-receptors in vivo extends beyond synaptic GABAergic neurotransmission and may, in the cerebellum, involve taurine as a ligand.


Subject(s)
Brain Chemistry/physiology , Purkinje Cells/chemistry , RNA Splicing/physiology , Receptors, GABA-B/analysis , Receptors, GABA-B/genetics , Animals , Azides/pharmacology , Blotting, Western , Brain/growth & development , Cell Membrane/chemistry , Cell Membrane/ultrastructure , Dendrites/chemistry , Dendrites/ultrastructure , GABA Antagonists/pharmacology , Guinea Pigs , Microscopy, Electron , Organophosphorus Compounds/pharmacology , Purkinje Cells/physiology , Purkinje Cells/ultrastructure , Rabbits , Rats , Rats, Sprague-Dawley , Receptors, GABA-B/metabolism , Synapses/chemistry , Synapses/ultrastructure , Synaptic Transmission/drug effects , Synaptic Transmission/physiology , Taurine/metabolism , gamma-Aminobutyric Acid/physiology
19.
J Biol Chem ; 273(18): 11158-63, 1998 May 01.
Article in English | MEDLINE | ID: mdl-9556603

ABSTRACT

The binding site for the co-agonist glycine on N-methyl-D-aspartate (NMDA) receptors has been mapped to the NR1 subunit whereas binding of the principal agonist glutamate is mediated by the NR2 subunits. Using the novel glycine site antagonist and photoaffinity label CGP 61594, distinct contributions of the NR2 subunit variants to the glycine antagonist binding domains of NMDA receptor subtypes are demonstrated. High affinity sites for CGP 61594 were exclusively displayed by NR1/2B receptors, as shown by their co-distribution with the NR2B subunit, by subunit-selective immunoprecipitation and by functional analysis of NR1/2B receptors expressed in Xenopus oocytes (inhibitory potency, IC50 = 45 +/- 11 nM). Other NMDA receptor subtypes are clearly distinguished by reduced inhibitory potencies for CGP 61594, being low for NR1/2A and NR1/2D receptors (IC50 = 430 +/- 105 nM and 340 +/- 61 nM, respectively) and intermediate for NR1/2C receptors (IC50 = 164 +/- 27 nM). Glycine antagonist sites with low and intermediate affinity for [3H]CGP 61594 were detected also in situ by radioligand binding in brain areas predominantly expressing the NR2A and NR2C subunits, respectively. Thus, [3H]CGP 61594 is the first antagonist radioligand that reliably distinguishes the glycine site of NMDA receptor subtypes. [3H]CGP 61594 is a promising tool to identify the NR2 subunit domains that contribute to differential glycine antagonist sites of NMDA receptor subtypes.


Subject(s)
Azides/metabolism , Glycine/metabolism , Quinolines/metabolism , Receptors, N-Methyl-D-Aspartate/antagonists & inhibitors , Allosteric Regulation , Azides/pharmacology , Binding Sites , Cell Line , Excitatory Amino Acid Antagonists/metabolism , Excitatory Amino Acid Antagonists/pharmacology , Humans , Precipitin Tests , Quinolines/pharmacology , Receptors, N-Methyl-D-Aspartate/classification , Receptors, N-Methyl-D-Aspartate/metabolism , Recombinant Proteins/metabolism , Tritium
20.
Life Sci ; 62(17-18): 1611-5, 1998.
Article in English | MEDLINE | ID: mdl-9585145

ABSTRACT

The rules governing the assembly of GABA(A) receptors in vivo were assessed in subunit mutant mice. The transcription of individual subunit genes was regulated independently. The lack of a particular subunit did not result in a molecular rescue by an enhanced transcription of other subunits. In addition, the availability of an alpha- and beta-subunit was essential for receptor formation. Finally, highly selective recognition processes directed the subcellular targeting of receptors. The loss of a particular receptor subtype (alpha5) did not lead to a subcellular redistribution of the remaining subtype (alpha2) present in the same cell.


Subject(s)
Receptors, GABA-A/biosynthesis , Animals , Gene Expression Regulation , Mice , Mice, Mutant Strains , Protein Processing, Post-Translational , Receptors, GABA-A/classification , Receptors, GABA-A/genetics
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